AimsTo search for culturable Burkholderia species associated with annual ryegrass in soils from natural pastures in Portugal, with plant growth-promoting effects. Methods and ResultsAnnual ryegrass seedlings were used to trap Burkholderia from two different soils in laboratory conditions. A combined approach using genomic fingerprinting and sequencing of 16S rRNA and recA genes resulted in the identification of Burkholderia strains belonging to the species Burkholderia graminis, Burkholderia fungorum and the Burkholderia cepacia complex. Most strains were able to solubilize mineral phosphate and to synthesize indole acetic acid; some of them could produce siderophores and antagonize the phytopathogenic oomycete, Phytophthora cinnamomi. A strain (G2Bd5) of B.graminis was selected for gnotobiotic plant inoculation experiments. The main effects were the stimulation of root growth and enhancement of leaf lipid synthesis and turnover. Fluorescence insitu hybridization and confocal laser microscopy evidenced that strain G2Bd5 is a rhizospheric and endophytic colonizer of annual ryegrass. ConclusionsThis work revealed that annual ryegrass can naturally associate with members of the genus Burkholderia. A novel plant growth promoting strain of B.graminis was obtained. Significance and Impact of the StudyThe novel strain belongs to the plant-associated Burkholderia cluster and is a promising candidate for exploitation as plant inoculant in field conditions.